Functional characterization of the internal ribosome entry site of eIF4G mRNA.
The eIF4 group initiation factors are required for cap-dependent translation initiation. Infection of mammalian cells by picornaviruses results in proteolytic cleavage of one of these factors, eIF4G, which severely restricts cap-dependent initiation but permits cap-independent initiation to proceed from an internal ribosome entry site (IRES) in picornaviral RNAs. The first 357 nucleotides (nt) of the 5'-untranslated region of eIF4G mRNA also contains an IRES. Using bicistronic constructs for expression in K562 cells, we have now shown that progressive deletions of the 5'-untranslated region can have either stimulatory or inhibitory effects. Furthermore, a 101-nt segment exhibits full IRES activity, and an 81-nt segment exhibits detectable IRES activity. A polypyrimidine tract (PPT) at the 3' terminus is essential for internal initiation, a property which is characteristic of picornaviral IRESs but not the other host cellular IRESs studied to date. IRES activity does not require sequences beyond 357 nt. Out-of-frame AUGs have no effect on IRES-driven luciferase expression when introduced upstream of the PPT but markedly decrease expression when introduced at sites between the PPT and the authentic initiation codon at nt 369. These results suggest that the ribosomal subunit enters at or near the PPT and then scans downstream for the initiation codon.
Gan
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Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, Louisiana 71130-3932, USA.
LaCelle
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Rhoads
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D018387:Codon, Initiator; D039603:Eukaryotic Initiation Factor-4G; D005786:Gene Expression Regulation; D005796:Genes; D017930:Genes, Reporter; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000096:biosynthesis* / Q000235:genetics; D011743:Pyrimidines; D012313:RNA; D012333:RNA, Messenger / Q000235:genetics*; D016364:Reading Frames; D012270:Ribosomes / Q000378:metabolism*; D017384:Sequence Deletion
D018387:Codon, Initiator; D039603:Eukaryotic Initiation Factor-4G; D010448:Peptide Initiation Factors; D011743:Pyrimidines; D012333:RNA, Messenger; C039919:RNA, recombinant; D012313:RNA